Iron-sulfur clusters containing a Fe2(mu2-S)2 core ([2Fe-2S] clusters) are one of the most ubiquitous electron carriers in nature and are integral components of Complex I, II, and III of the mitochondrial respiratory chain. Recent studies have also implicated the involvement of (2Fe-2S] clusters in iron-sulfur cluster assembly, iron insertion into porphyrin to form hemes, the synthesis of biotin from dethiobiotin, proton translocation, regulatory processes in response to oxidative stress and dehydratase reactions, although the precise role of the [2Fe-2S] clusters in these important biological processes has yet to be fully defined. The long-term goal of this research is a detailed understanding at the molecular level of the physicochemical properties of biological (2Fe-2S] centers with particular emphasis on the factors determining midpoint potential and the origin of their apparent functional diversity. Ultimately this will lead to enhanced understanding of the origin of human protoporphyria and diseases associated with inhibition of the respiratory chain enzymes. The approach involves using biophysical spectroscopic techniques, electron paramagnetic resonance, UV/visible/near-IR absorption, circular dichroism and magnetically induced circular dichroism, and resonance Raman, to investigate the electronic, magnetic and structural properties of [2Fe-2S] centers in a wide range of native and mutated enzymes and proteins. These include human and cyanobacterial ferredoxins, human ferrochelatase, spinach dihydroxyacid dehydratase, Escherichia coil biotin synthase, the Azotobacter vinelandii nifU gene product. In addition, enzymes and proteins extracted from or closely related to Complex I, II and III of the mitochondrial respiratory chain will be investigated, i.e. individual subunits of Paracoccus denitrificans NADH dehydrogenase, Escherichia coli fumarate reductase and a soluble Rieske protein fragment isolated from bovine heart mitochondria.